1. Field of the Invention
The present invention relates to fluorescent labels for various proteinaceous species and more particularly to fluorescent labels useful for the preparation of immunoreagents comprising fluorescently labeled antigens or antibodies.
2. Description of Related Art
Immunoassay is a field where sensitivity is of prime importance due to the low analyte levels that are measured. Radioimmunoassay sensitivity is limited to 10.sup.-12 M and is more often only in the 10.sup.-8 to 10.sup.-10 M range. In addition, radiolabels suffer from the drawbacks of short half life and handling hazards.
The sensitivity of fluorescence assays, although theoretically very high, is limited by the presence of background fluorescence. In many situations, it is impossible to reduce the background sufficiently (by appropriate filtration and other techniques known in the art) to obtain the desired sensitivity.
Time resolution offers an independent means of isolating the specific fluorescent signal of interest from nonspecific background fluorescence. This can be done if the label has much longer-lived fluorescence than the background, and if the system is illuminated by an intermittent light source such that the long-lived label can be measured during the dark period subsequent to the decay of the short-lived background. Such techniques are described in greater detail in German Offenlegungschrift No. 2,628,158 published Dec. 30, 1976.
The long-lived fluorescence (0.1-5 msec) of the aromatic diketone chelates of certain rare earth metals, for example, europiumbenzoylacetonate and europiumbenzoyltrifluoracetonate, has been known for some time. The chelating agent absorbs light and transfers it to the metal ion, which fluoresces. German OLS No. 2,628,158 describes the use of time resolution in fluorometric immunoassays (FIA) through the use of fluorescent labels whose emissions are long lived as compared to those of species which produce background interferences in such assays. This publication also provides a useful discussion of the techniques of FIA and its advantage over other immunoassay techniques such as radioimmunoassay (RIA).
The fluorescent immunoreagents described in German OLS No. 2,628,158 comprise at least one member of the immune-complement system, i.e., an antibody or an antigen, "conjugated" with a rare earth chelate. Such "conjugation" can be achieved in one of two ways:
(1) by first labeling, i.e., attaching the rare earth chelate to the antigen as described in "Fluorescent Antibody Techniques and Their Application" by A. Kawamura, Ed., University Park Press, Baltimore, Maryland, 1969, and then adding antibody to the conjugated antigen whereby the antibody and antigen join in the usual fashion, or:
(2) by covalent bonding of the antibody to the chelate via a chemical group which binds to both antibodies and the chelates.
The problem with immunoreagents of the type described in German OLS No. 2,628,158 is that the fluorescent labeling species, namely the rare-earth chelates, are quenched, i.e., their fluorescence is extinguished, when contacted with water. This problem, hereinafter referred to as an "aqueous stability" problem, is particularly serious because a principal use for fluorescent labeled immunoreagents is in the assay of aqueous biological liquids such as blood, serum, etc. If aqueous stability could be conferred on these materials, they would be useful as fluorescent labels for these biological liquids, thus allowing increased fluorescence immunoassay sensitivity by the use of time resolution of signal from background.
Belgian Patent Publication No. 843,647 published Dec. 30, 1976, describes scintillation counting compositions comprising polymeric particles derived from a latex and loaded with at least one hydrophobic fluor as well as methods for preparing such latices.
Leif, R. C. et al, Clinical Chemistry, Vol. 23, No. 8 (1977) suggest techniques for stabilizing rare earth chelates for use in aqueous liquids. The method generally comprises attaching a chelating agent to the surface of a polymeric bead and then chelating several other chelating agents and the bead-attached chelating agent to a single rare earth atom.
U.S. Pat. No. 3,853,987 issued Sept. 1, 1971, suggests the incorporation of "tracer" molecules throughout the volume of particles of acrylic acid derivatives in a latex and use of such particles as labels for immunoreagents. Although this patent teaches that the tracer is incorporated throughout the particle volume, there is no teaching of how the "tracer", fluorescent or radioactive, is incorporated into the particle. The only teachings are to attaching such "tracers" to acrylic polymers or adhering the "tracers" to the surface of such polymers.